Mechanisms of Klebsiella pneumoniae gastrointestinal colonization

肺炎克雷伯菌胃肠道定植机制

• 批准号: 10736879
• 负责人: Muhammad Ammar Zafar
• 金额: $ 55.83万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-14 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10736879
• 关键词: Affect; Animal Model; Antibiotic Resistance; Arginine; Biological; Biological Assay; Bypass; Catabolism; Cell membrane; Cellular Membrane; Data; Development; Diet; Disease; Ethanolamines; Failure; Feces; Gastrointestinal tract structure; Genes; Genetic Screening; Gnotobiotic; Gram-Negative Bacteria; Immune system; Immunocompetent; In Vitro; Infection; Inflammatory; Investigation; Kinetics; Klebsiella pneumoniae; Knock-out; Knowledge; Libraries; Link; Metabolic Pathway; Metabolism; Metagenomics; Modeling; Molecular; Mucous Membrane; Multi-Drug Resistance; Mus; Nosocomial Infections; Nutrient; Nutritional; Operon; Oral; Oropharyngeal; Outcome; Pathway interactions; Patients; Prevention; Process; Regulation; Role; Route; Site; Source; System; burden of illness; colonization resistance; combat; diarrheal disease; digital; drug resistant pathogen; enteric pathogen; epidemiology study; fitness; gastrointestinal; gene product; genomic locus; gut colonization; gut microbiome; gut microbiota; host colonization; in vivo; insight; member; metabolomics; metagenome; metagenomic sequencing; microbiome; microbiome components; microbiota; microorganism; mouse model; mutant; novel; pathogen; pathogenic bacteria; public health relevance; success; transmission process; uptake

Abstract Hospital-acquired infections (HAI) resulting from the transmission of drug-resistant pathogens affect hundreds of millions of patients worldwide. Klebsiella pneumoniae (Kpn), a gram-negative bacterium, is notorious for causing HAI, with many of these infections difficult to treat as Kpn has become multi-drug resistant. Epidemiological studies suggest that gastrointestinal (GI) colonization of Kpn is a major reservoir through which Kpn can cause disease manifestations either in the colonized host or transmit from host to host. This site of Kpn colonization has not been the focus of previous studies as a tractable model of Kpn GI colonization, and host-to-host transmission did not exist. We have recently developed a murine model that allows for the study of Kpn mucosal (oropharynx and GI) colonization, shedding within feces, and transmission through the fecal-oral route. Using an oral route of inoculation and fecal shedding as a marker for GI colonization, we show that Kpn can asymptomatically colonize the GI tract of immunocompetent mice and modifies the host GI microbiota. We premise that specific Kpn genes contribute to its GI colonization, and the products of these genes could serve as novel targets for the prevention of the establishment of GI colonization. More recently, we used our murine model to screen a library of Kpn random transposon mutants (In-seq) to identify the complete set of “GI colonization” genes from a single isolate. A metagenomics sequencing analysis further identified bacterial species and the metabolic pathways affected by Kpn in the GI tract. Herein, we will focus on two sets of pathways identified through In-seq whose products allow Kpn to overcome colonization resistance provided by the resident gut microbiota. Thus, in Aim#1, we will focus on the ethanolamine utilization pathway genes (eut) of Kpn that allow it to utilize ethanolamine (EA), a byproduct of cellular membranes and diet in the gut that can serve as an alternative nutrient source. Unlike many other enteric pathogens that contain a single eut operon, Kpn has two genetically distinct eut operons. We will identify the role of each eut locus in EA metabolism and determine the underlying molecular mechanism through which EA metabolism provides Kpn with a fitness advantage against members of the microbiome. Aim#2 will take a different approach by focusing on the contact-dependent killing machinery of the Kpn (Type 6 secretion system [T6SS]) in overcoming colonization resistance provided by the resident microbiota. We will focus on the unique regulatory mechanism that modulates the expression of Kpn T6SS in the GI tract and provide it with a selective and competitive advantage against the resident gut microbiota. Results from these studies would provide us with a fundamental understanding of the molecular mechanisms involved in the establishment of GI colonization by an incoming pathogen. These studies will also lay the groundwork for developing potential strategies to reduce the Kpn disease burden.

摘要 由耐药病原体传播引起的医院获得性感染（HAI）影响数百人， 全世界数百万患者。肺炎克雷伯氏菌（Kpn）是一种革兰氏阴性菌， 导致HAI，其中许多感染难以治疗，因为Kpn已成为多药耐药。 流行病学研究表明，胃肠道（GI）定植的Kpn是一个主要的水库，通过 该Kpn可在定殖宿主中引起疾病表现或在宿主间传播。本网站 Kpn定殖并不是以前研究的重点，因为Kpn GI定殖的易处理模型， 主机到主机传输不存在。我们最近开发了一种小鼠模型， Kpn粘膜（口咽和GI）定植、粪便内脱落和经粪-口传播 路线使用口服接种途径和粪便脱落作为GI定植的标志，我们发现 Kpn可以无症状地定殖于免疫活性小鼠的胃肠道，并改变宿主的胃肠道 微生物群我们假设特定的Kpn基因有助于其GI定植，并且这些基因的产物 可以作为新的目标，防止建立GI殖民。最近，我们使用 我们的鼠模型筛选Kpn随机转座子突变体文库（In-seq），以鉴定完整的Kpn转座子突变体组。 来自单个分离物的“GI定植”基因。宏基因组测序分析进一步鉴定了细菌 种属和胃肠道中受Kpn影响的代谢途径。在此，我们将重点介绍两组途径 通过In-seq识别，其产品允许Kpn克服居民提供的殖民抵抗力 肠道微生物群因此，在目标#1中，我们将关注Kpn的乙醇胺利用途径基因（eut）， 允许它利用乙醇胺（EA），这是肠道中细胞膜和饮食的副产品，可以作为一种 替代营养源。与许多其他含有单个eut操纵子的肠道病原体不同，Kpn具有两个eut操纵子。 遗传上不同的eut操纵子。我们将确定每个eut基因座在EA代谢中的作用并确定 潜在的分子机制，通过它EA代谢提供Kpn与健身优势， 微生物组的成员。目标2将采取不同的方法，重点关注接触依赖性杀伤 Kpn（6型分泌系统[T6 SS]）的机制，以克服殖民抵抗提供的 常驻微生物群我们将集中在独特的调节机制，调节表达 的Kpn T6 SS在胃肠道，并提供了一个选择性和竞争优势，对居民肠道 微生物群这些研究的结果将为我们提供对分子生物学的基本理解。 通过引入的病原体建立GI定殖的机制。这些研究还将 为制定减少Kpn疾病负担的潜在战略奠定基础。